The objective of this grant is to continue studies on the structure, function and transcription regulation of both constitutively expressed and xenobiotic inducible hepatic mitochondrial cytoChrome P-450. The work carried out during the current grant period has identified a potentially new mechanism of protein transport into mitochondria that involves a proteolytic cleavage of the ER membrane associated P-4501A1 immediately past the transmembrane domain of the protein. The 5' truncated, and mitochondrial imported P-450 accepts electrons through ferredoxin and demonstrates catalytic properties similar to mitochondrial P-450mt2. Additionally, a female predominant P-450 involved in cholesterol C-27 hydroxylation and vitamin D 25-hydroxylation (CYP27) has been shown to be regulated by pituitary steroids and growth hormone (GH). Putative growth hormone response region of the promoter that binds to specific nuclear protein(s) has been characterized. Based on this, the broad objective is to continue studies on both of these aspects as follows: 1) Further characterization of mechanisms of N-terminal cleavage of P- 4501A1 to expose the amphiphilic helical region that acts as a potential mitochondrial signal sequence. A combination of site directed mutagenesis and in vivo/in vitro protein transport will be used to further elucidate the mechanism of "protein hopping" from ER to mitochondria. The generality of this mechanism will be tested with other P-450 and non P-450 proteins. 2) The endopeptidase responsible for the release of ER bound P-4501A1 will be characterized, and if possible purified to gain further insight on this new transport mechanism. 3) The structure and function of the truncated P-4501A1 imported into mitochondria will be studied by in vitro reconstitution of purified enzyme using varied substrates and electron transfer proteins, and the ferredoxin binding region will be mapped by a combination of chemical cross-linking and in vitro mutagenesis. 4) The mechanisms of GH regulation of CYP27 gene will be studied by a combination of in vivo transcription analysis, characterization of proteins binding to the putative GH responsive site, and study the phosphorylation pattern to gain insight on the 6H mediated activation mechanism. 5) Characterize the dual transcription promoters of the mouse CYP27 gene and determine tissue specific differences in the promoter site selection to understand their functional and physiological significance.